Our research is in the general area of molecular genetics and deals specifically with the structure and function of eukaryotic genes and chromosomes. The long range objectives are to define the structural organization of the centromeres and the telomeric regions of eukaryotic chromosomes, and to understand the molecular mechanims of chromosome segregation in mitotic and meiotic cell divisions. We rely in part on the use of the yeast, Saccharomyces cerevisiae, as an experimental system, because molecular cloning technology coupled with a DNA transformation system can be used to isolate and study functional centromeric DNA from this organism. Thus, we are able to construct in vitro centromere-containing plasmids that replicate and segregate properly through mitosis and meiosis in the yeast cell. These minichromosomes constitute an invaluable probe for the study of chromosome structure and function. Specific aims of the propose research include: (1) studies on centromete structure, including the nucleotide sequences of centromere DNAs and their nucleosomal organization; (2) isolation and characterization of centromere binding proteins; (3) studies on the role of centromere binding proteins in cell division and chromosome segregation; (4) investigation of the molecular interaction between centromere DNA, centromere binding proteins, and tubulin and/or microtubules; (5) functional studies on minichromosomes, including the mechanism of meiotic pairing and segregation, centromere-replicator interactions and copy number control; (6) studies on linear minichromosomes and the isolation and structural properties of yeast telomeric DNAs; and (7) comparative studies on centromeres and centromere binding proteins from yeast and other eukaryotes. Studies on the molecular mechanisms of cell division and chromosome segregation in yeast will offer an approach to the understanding of the mechanisms as they function normally and abnormally in higher eukaryotes.